Salinity Stress Ameliorates Pigments, Minerals, Polyphenolic Profiles, and Antiradical Capacity in Lalshak

Previous studies have shown that salinity eustress enhances the nutritional and bioactive compounds and antiradical capacity (ARC) of vegetables and increases the food values for nourishing human diets. Amaranth is a salinity-resistant, rapidly grown C4 leafy vegetable with diverse variability and usage. It has a high possibility to enhance nutritional and bioactive compounds and ARC by the application of salinity eustress. Hence, the present study aimed to evaluate the effects of sodium chloride stress response in a selected Lalshak (A. gangeticus) genotype on minerals, ascorbic acid (AsA), Folin–Ciocalteu reducing capacity, beta-carotene (BC), total flavonoids (TF), pigments, polyphenolic profiles, and ARC. A high-yield, high-ARC genotype (LS6) was grown under conditions of 0, 25, 50, and 100 mM sodium chloride in four replicates following a block design with complete randomization. We recognized nine copious polyphenolic compounds in this accession for the first time. Minerals, Folin–Ciocalteu reducing capacity, AsA, BC, pigments, polyphenolic profiles, and ARC of Lalshak were augmented progressively in the order: 0 < 25 < 50 < 100 mM sodium chloride. At 50 mM and 100 mM salt concentrations, minerals, AsA, Folin–Ciocalteu reducing capacity, BC, TF, pigments, polyphenolic profiles, and ARC of Lalshak were much greater than those of the control. Lalshak could be used as valuable food for human diets as a potent antioxidant. Sodium chloride-enriched Lalshak provided outstanding quality to the final product in terms of minerals, AsA, Folin–Ciocalteu reducing capacity, BC, TF, pigments, polyphenolic profiles, and ARC. We can cultivate it as a promising alternative crop in salinity-prone areas of the world.


Introduction
Consumers' acceptability largely depends on the color, flavor, and taste of products. Presently, coloring food products has attracted the favor of consumers. These products have received much interest from consumers in aesthetic, nutritional, and safety aspects of food. The demand for natural pigments such as betacyanins, betaxanthins, betalains, anthocyanin, amaranthine, carotenoids, and chlorophylls is increasing considerably day by day. The selected Lalshak genotype is bright red-violet due to the presence of abundant betalains. Leaves of amaranths are an exclusive source of betalains (betaxanthins and betacyanins) that have a strong antiradical capacity (ARC) [1]. Betalains could be used as a food colorant in low-acid foods as they have higher pH stability than anthocyanins [2].

Determination of Chlorophylls and Carotenoids
The Lalshak leaves were extracted in 80% acetone to estimate total chlorophyll, chlorophyll b, carotenoids, and chlorophyll a [64][65][66][67]. A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to read the absorbance at 663 nm for chlorophyll a, 646 nm for chlorophyll b, and 470 nm for carotenoids. Data were expressed as µg chlorophyll per g fresh weight (FW) and mg carotenoids per 100 g FW.

Betacyanins and Betaxanthins Content Measurement
The Lalshak leaves were extracted in 80% methyl alcohol containing 50 mM ascorbate to measure betacyanins and betaxanthins [68][69][70]. A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to measure the absorbance at 540 nm for betacyanins and 475 nm for betaxanthins. The results were expressed as the nanogram betanin equivalent per gram FW for betacyanins and nanogram indicaxanthin equivalent per gram FW for betaxanthins.

Estimation of BC
Fresh leaves (500 mg) were thoroughly mixed with 80% acetone (10 mL) using a mortar and pestle. We determined BC by centrifuging the mixture for 3-4 min at 10,000× g [71][72][73][74]. We separated the filtrate in a flask and maintained the final volume of 20 mL. We measured the absorbance using a spectrophotometer (Tokyo, Japan) at 480 and 510 nm. Finally, we calculated BC as mg 100 g −1 FW.

Estimation of AsA
AsA and DHA were determined from fresh leaves. DHA was reduced to AsA by pre-incubating the sample using dithiothreitol (DTT). Fe 3+ was converted to Fe 2+ with the reduction of AsA. Fe 2+ complexes were formed by reacting Fe 2+ and 2, 2-dipyridyl [75,76]. We took the optical density of the complexes using a Hitachi spectrophotometer (Tokyo, Japan) at 525 nm. Finally, we calculated AsA as mg 100 g −1 FW.

Sample Extraction and Determination of Folin-Ciocalteu Reducing Capacity, TF, and ARC
To avoid direct sunshine, we dried leaves in a shady place. We extracted both the ground dried and fresh leaves (30 d) separately with a mortar and pestle. Folin-Ciocalteu reducing capacity was estimated from fresh leaves, whereas ARC and TF contents were estimated from dried leaves. Exactly 0.25 g samples were combined with 10 mL MeOH (90%) in a tightly capped bottle. We placed the mixture in a shaker (Tokyo, Japan) at 60 • C for 1 h. For Folin-Ciocalteu reducing capacity, ARC, and TF estimation, we stored the final filtrate until use. Folin-Ciocalteu reducing capacity and TF were estimated by the Folin-Ciocalteu reagent and AlCl 3 colorimetric methods, respectively [77]. We used a spectrophotometer (Hitachi, Tokyo, Japan) to read the absorbance at 415 and 760 nm. Folin-Ciocalteu reducing capacity and TF were measured using gallic acid and rutin standard curves as gallic acid and rutin equivalent µg GAE g −1 of FW and µg RE g −1 DW. The ARC was estimated by radical degradation by DPPH and ABTS + assay [78,79]. We measured the inhibition % of ABTS + and DPPH equivalent to the control using the equation: where Ab represents the blank sample absorbance (10 µL and 150 µL MeOH for ARC (ABTS) and DPPH, respectively, as a substitute of leaf extract), and AS is the absorbance of the sample. Finally, we calculated ARC as µg Trolox equivalent g −1 DW.

Sample Extraction and Determination of Polyphenolic Profiles by HPLC
Exactly 1 g of fresh leaves was extracted in 10 mL MeOH (80%) comprising ascorbate (1%). We homogenized the mixture thoroughly and placed it in a 50 mL test tube (tightly capped). Then, we placed the test tube in a shaker (Scientific Industries Inc., New York, NY, USA) at 400 rpm for 15 h. The mixture was filtered using a 0.45 µm filter (Springfield, MA, USA) and centrifuged at 10,000× g for 15 min. We estimated polyphenolic compounds from the filtrate. We repeated all extractions 3 times. For the HPLC determination of polyphenolic compounds, we followed the method of Sarker and Oba [50]. We equipped Shimadzu HPLC equipment (Kyoto, Japan) with a degasser, detector, and binary pump. A CTO-10 AC (STR ODS-II, 150 × 4.6 mm, 5 µm; Shinwa Chemical Industries, Ltd., Kyoto, Japan) column was used for the separation of polyphenolic compounds. We pumped Solvent A (acetic acid 6% (v/v) in water) and Solvent B (acetonitrile) at 1 mL/min for 70 min. We used a gradient program to run the HPLC system with 0-15%, 15-30%, 30-50%, and 50-100% acetonitrile for 45, 15, 5, and 5 min. We maintained the column temperature of 35 • C with an injection volume of 10 µL. We set a Shimadzu SPD-10Avp UV-vis detector at 280, 360, and 370 nm to continuously monitor polyphenolic compounds. We identified the compound by comparing the retention time and UV-vis spectra with their respective standards. Finally, we calculated polyphenolic compounds as µg g −1 FW.

Quantification of Polyphenolic Compounds
We quantified each polyphenolic compound using the corresponding standards of calibration curves. We prepared stock solutions (100 mg/mL) by dissolving 9 polyphenolic compounds with 80%MeOH. We quantified polyphenolic compounds using standard curves (10,20,40,60,80, and 100 µg/mL) with external standards. Co-chromatography of samples' retention times spiked with commercially available standards. We identified and matched the polyphenolic compounds utilizing UV spectral characteristics.

Statistical Analysis
To obtain a replication mean, we averaged each treatment from all the sample data of a trait [80][81][82][83]. We biometrically and statistically analyzed the mean data of various traits [84][85][86][87]. Statistix 8 software was used to analyze the data to obtain an analysis of variance (ANOVA) [88][89][90]. Duncan's multiple range test (DMRT) at a 1% level of probability was used to compare the means. The results were reported as the mean ± SD of four separate replicates.
traits [84][85][86][87]. Statistix 8 software was used to analyze the data to obtain an analysis variance (ANOVA) [88][89][90]. Duncan's multiple range test (DMRT) at a 1% level of pro bility was used to compare the means. The results were reported as the mean ± SD of fo separate replicates.

Influence of Sodium Chloride Stress on Color Parameters and Pigments
Figures 1 and 2 represent the color parameters and pigments under different sodiu chloride stresses. Preference, choice making, and acceptability of the product mostly d pend on leaf color, which contributes significantly to the choice of consumers. It is a k indicator for evaluating the ARC of leafy vegetables [91]. The LS6 accession had high p itive a* and b* values, indicating the presence of abundant red and yellow color pigme (betaxanthins, carotenoids, betacyanins, anthocyanins, and betalains). Our obtained sults corroborated the results of Colonna et al. [91]. Betacyanins, chroma, L*, carotenoi betaxanthins, betalains, a*, and b* values were progressively augmented in the ord Control < 25 < 50 < 100 mM salt stress. In contrast, total chlorophyll, chlorophyll b, a chlorophyll a content were drastically reduced in the order: Control > 25 > 50 > 100 m salt stress. Carotenoids, L*, chroma, b*, betacyanins, betaxanthins, betalains, and a* w augmented by 14%, 0%, 1%, 1%, 2%, 4%, 1%, 4%; 28%, 32%, 3%, 7%, 7%, 6%, 5%, 3%, 11 and 59%, 6%, 14%, 16%, 10% 10%, 8%, 22% under 25; 50; and 100 mM salt concentratio respectively. In contrast, chlorophyll b, chlorophyll a, and total chlorophyll content w reduced by 2%, 6%, 4%; 9%, 9%, and 9%; and 17%, 19%, 18%, respectively, compared control conditions ( Figure 3). Petropoulos et al. [52] reported that the chlorophylls Cichorium spinosum were drastically reduced with an increment in sodium chloride stre    Sodium chloride stress affected plant growth and development through decreasin stomatal conductivity, which restricts CO2 influx to leaves and causes osmotic stress plants, reduction in water potential, and unfavorable CO2/O2 ratios in chloroplasts, redu ing photosynthesis. Lim et al. [53] reported that different salt concentrations augmente   Sodium chloride stress affected plant growth and development through decreasing stomatal conductivity, which restricts CO2 influx to leaves and causes osmotic stress in plants, reduction in water potential, and unfavorable CO2/O2 ratios in chloroplasts, reducing photosynthesis. Lim et al. [53] reported that different salt concentrations augmented carotenoid content. They observed the highest increment (two-fold) in carotenoids under 50 and 100 mM salt concentrations in comparison to the control conditions. Alam et al.  Sodium chloride stress affected plant growth and development through decreasing stomatal conductivity, which restricts CO 2 influx to leaves and causes osmotic stress in plants, reduction in water potential, and unfavorable CO 2 /O 2 ratios in chloroplasts, reducing photosynthesis. Lim et al. [53] reported that different salt concentrations augmented carotenoid content. They observed the highest increment (two-fold) in carotenoids under 50 and 100 mM salt concentrations in comparison to the control conditions. Alam et al. [54] observed both stimulation and reduction in carotenoid content in saltstressed purslane. To regulate plant development under sodium chloride stress, the plant accelerates the mevalonic acid pathway for the biogenesis of abscisic acid from carotenoids. Thus, sodium chloride stress enhances the synthesis of carotenoids to accelerate the mevalonic acid pathway [47]. The decline in pigment for photosynthesis under salt stress is also linked with the oxidation of chlorophyll pigment through free radicals, interference of salt ions with pigment-protein complexes [92], chloroplast disruption, and increased activity of chlorophyllase enzymes responsible for the degradation of chlorophylls [93]. The presence of betalain pigments (betaxanthin and betacyanin) may act as an antioxidant and absorb radiation significantly to protect against excessive harmful light in the chloroplasts. These findings were corroborative to the findings of Jain et al. [94]. In Disphyma australe, they reported that salt-induced plants with increased betalains exhibited more tolerant physiology through the production of less H 2 O 2 , faster recoveries of PSII, and increased rates of assimilation of CO 2 , and photochemical quenching, photochemical quantum yields, and water-use efficiencies. Moreover, betalains (betacyanins and betaxanthins) protect the chloroplasts from salinity stress by scavenging reactive oxygen species in thylakoids [95] and through faster recoveries of PSII, photochemical quenching, and photochemical quantum yields [94].

Sodium Chloride Impact on Minerals (Macroelements and Microelements)
Macroelements and microelements in Lalshak are presented in Figures 4 and 5. The studied Lalshak demonstrated copious macroelements and microelements, which corroborated with the results of Shukla et al. [96], who reported very high levels of minerals in open-field-grown A. tricolor. Lalshak has greater iron and zinc compared to the leaves of cassava [97] and beach peas [98]. The previous study showed copious amounts of Mn, Fe, Cu, and Zn in different A. spp. [99]. They demonstrated greater levels of copper and iron in different A. spp., which were superior to kale, and Zn levels of different A. spp. were also superior to spinach, kale, and black nightshade. At 100 mM salt concentration, the maximum calcium, magnesium, sulfur, iron, manganese, copper, zinc, sodium, molybdenum, and boron contents were noted, while at control conditions, the minimum calcium, magnesium, manganese, zinc, sodium, and boron contents were displayed. Similarly, under control and 25 mM salt stress conditions, the minimum sulfur, iron, copper, and molybdenum contents were detected. Calcium, magnesium, manganese, zinc, sodium, and boron contents were gradually increased in the order: Control < 25 < 50 < 100 mM salt concentrations. Inversely, potassium and phosphorus contents extremely declined in the order: Control > 25 > 50 > 100 mM salt concentrations.

Influence of Sodium Chloride on Phytochemicals
The Folin-Ciocalteu reducing capacity, BC, AsA, TF, and ARC varied noticeably at different sodium chloride concentrations (Figure 7). cytosolic Na + levels appropriately [103]. In many plant species, the main physiological mechanism of salt tolerance is the uptake of selective K + against Na + [104].

Response of Sodium Chloride Stress on Polyphenolic Compounds
The HPLC-identified polyphenolic profile values of Lalshak (accession LS6) under four salt stresses were collated with polyphenolic compounds using the respective peaks of the compounds (Table 1). Figure 9 designates the identified polyphenolic profiles of the Lalshak genotype under four salt stresses. Nine polyphenolic profiles including six flavonols, namely quercetin, rutin, iso-quercetin, hyperoside, kaempferol, and myricetin; one flavanol (catechin); one flavone (apigenin); and one flavanone (naringenin) were identified in adequate quantities in Lalshak leaves. We identified six polyphenolic compounds (iso-quercetin, kaempferol, myricetin, catechin, apigenin, and naringenin) for the first time in this genotype. Across polyphenolic profiles, rutin is the most preponderant flavonoid compound in Lalshak followed by quercetin, naringenin, and myricetin ( Figure 9). Khanam et al. [107] and Khanam and Oba [108] reported three flavonoids (quercetin, rutin, and hyperoside) in amaranths. Abiotic stresses such as salinity generate various ROS, such as H 2 O 2 , superoxide, hydroxyl radical, singlet oxygen, etc., and cause oxidative damage in plants which, finally, can oxidize lipids, DNA, proteins, and various cellular macromolecules. To cope with oxidative damage, plants accumulate non-enzymatic antioxidant compounds, such as polyphenols, flavonoids, and antioxidant enzymes. Generally, the accumulation of polyphenols that possess antioxidant properties is stimulated in response to ROS increases under biotic and abiotic stresses. They are plentiful and present in plant tissues [109]. Polyphenols can chelate transition-metal ions, can directly scavenge molecular species of active oxygen, and may quench lipid peroxidation by trapping the lipid alkoxyl radical. Furthermore, flavonoids and phenylpropanoids are oxidized by peroxidase and act in the H 2 O 2 -scavenging, phenolic/AsA/POD system. Antioxidant activity is the combined results of all enzymatic and non-enzymatic antioxidant activity in natural and/or biotic/abiotic stress. Tolerant plant genotypes usually have a better antioxidant content to protect them from oxidative stress by maintaining high antioxidant enzyme and antioxidant molecule activities under stress conditions. Antioxidants protect the cells from free radicals and, therefore, have been considered as a method to improve plant defense responses [110]. Antioxidant activity has a crucial role in maintaining the balance between the production and scavenging of free radicals [111].  Abiotic stresses such as salinity generate various ROS, such as H2O2, superoxide, hydroxyl radical, singlet oxygen, etc., and cause oxidative damage in plants which, finally, can oxidize lipids, DNA, proteins, and various cellular macromolecules. To cope with oxidative damage, plants accumulate non-enzymatic antioxidant compounds, such as polyphenols, flavonoids, and antioxidant enzymes. Generally, the accumulation of polyphenols that possess antioxidant properties is stimulated in response to ROS increases under biotic and abiotic stresses. They are plentiful and present in plant tissues [109]. Polyphenols can chelate transition-metal ions, can directly scavenge molecular species of active oxygen, and may quench lipid peroxidation by trapping the lipid alkoxyl radical. Furthermore, flavonoids and phenylpropanoids are oxidized by peroxidase and act in the H2O2-  Salt stress progressively augmented all flavonoid compositions. At 100 mM salt stress, all flavonoid compounds showed maximum contents, while the lowest flavonoid contents were recorded from the control treatment. Quercetin, rutin, hyperoside, myricetin, and naringenin were progressively augmented in the following order: Control < 25 < 50 < 100 mM salt stress. From control to 100 mM salt stress conditions, quercetin, rutin, hyperoside, myricetin, and naringenin ranged from 7.35 to 18.63, 14.62 to 32.47, 3.35 to 7.36, 7.48 to 15.48, and 9.14 to 16.58 µg g −1 FW, respectively (Figure 9). From control to 100 mM salt concentration conditions, quercetin, rutin, hyperoside, myricetin, and naringenin were sharply and remarkably augmented by 16%, 110%, and 153%; 21%, 56%, and 112%; 19%, 95%, and 120%; 9%, 57%, and 107%; and 14%, 36%, and 81% ( Figure 10). 50 < 100 mM salt stress. From control to 100 mM salt stress conditions, quercetin, rutin, hyperoside, myricetin, and naringenin ranged from 7.35 to 18.63, 14.62 to 32.47, 3.35 to 7.36, 7.48 to 15.48, and 9.14 to 16.58 µg g −1 FW, respectively (Figure 9). From control to 100 mM salt concentration conditions, quercetin, rutin, hyperoside, myricetin, and naringenin were sharply and remarkably augmented by 16%, 110%, and 153%; 21%, 56%, and 112%; 19%, 95%, and 120%; 9%, 57%, and 107%; and 14%, 36%, and 81% ( Figure 10). Iso-quercetin did not augment between control and 25 mM salt stress conditions; however, when increasing salt concentration from 25 to 100 mM, this compound sharply increased with an increase in salt concentration with a range from 6.01 to 8.96 µg g −1 FW. Apigenin sharply increased from control to 50 mM salt stress conditions with a range from 6.37 to 7.97 µg g −1 FW. However, when increasing salt concentration from 50 to 100 mM, the apigenin concentration statistically remained constant. Kaempferol and catechin ranged from 7.88 to 10.86 and 2.88 to 5.36 µg g −1 FW. These two compounds had statistical similarity between the control and 25 mM salt stress conditions and between 50 and 100 mM salt stress conditions; however, these two compounds were remarkably augmented from the control condition or 25 to 50 or 100 mM salt stress conditions (82%) (Figures 9 and 10).

Rt (min)
Among the four groups of polyphenolic profiles, the flavonols group is the most plentiful in Lalshak compared to other groups, followed by flavanones. Polyphenolic groups in Lalshak were in the order: flavonols > flavanones > flavones > flavanols ( Figure 11). All polyphenolic portions were abruptly increased under salt stress. All polyphenolic portions displayed maximum concentrations under 100 mM salt concentrations, although the control had minimum polyphenolic portions. From control to 100 mM salt concentration, flavonols, flavones, flavanols, flavanones, and total polyphenols ranged from 46.66 to 93.76, 6.37 to 8.06, 2.88 to 5.36, 9.14 to 16.58, and 65.05 to 123.76 µg g −1 FW, respectively ( Figure 11).

The Coefficient of Correlation Study
The coefficient of correlation among BC, AsA, TF, Folin-Ciocalteu reducing capacity, ARC (DPPH), and ARC (ABTS + ) are shown in Table 2. BC showed significant associations with AsA, TF, Folin-Ciocalteu reducing capacity, ARC (DPPH), and ARC (ABTS + ). This indicated that the augmentation of BC is predominately related to the enhancement of AsA, TF, Folin-Ciocalteu reducing capacity, ARC (DPPH), and ARC (ABTS + ). Similarly, AsA exhibited a significant inter-relationship with TF, Folin-Ciocalteu reducing capacity, ARC (DPPH), and ARC (ABTS + ). Both BC and AsA had significant and strong contributions to the ARC of the genotype. TF, Folin-Ciocalteu reducing capacity, ARC (DPPH), and ARC (ABTS + ) were significantly correlated with each other. Gharibi et al. [112] observed a positive association among total Folin-Ciocalteu reducing capacity, TF, and ARC in Achillea species. Alam et al. [54] also reported a significant correlation among carotenoids, Folin-Ciocalteu reducing capacity, AsA, BC, and TF with ARC (FRAP) in salt-stressed purslane. Significant positive associations of AsA, Folin-Ciocalteu reducing capacity, BC, TF, ARC (DPPH), and ARC (ABTS + ) signifies the strong antioxidant potential of TF and Folin-Ciocalteu reducing capacity of the genotype. Likewise, significant positive correlations between ARC (DPPH) and ARC (ABTS + ) confirmed the validation of the antioxidant potential of the genotype by estimation of ARC using two different methods.

Conclusions
Sodium chloride stress remarkably augmented a*, calcium, L*, AsA, magnesium, b*, ARC (DPPH), sulfur, TF, iron, BC, manganese, ARC (ABTS + ) copper, zinc, sodium, Folin-Ciocalteu reducing capacity, molybdenum, boron, chroma, polyphenolic profiles, and pigments such as betacyanins, betaxanthins, betalains, and carotenoids of Lalshak leaves. All mineral contents, AsA, Folin-Ciocalteu reducing capacity, BC, TF, pigments, polyphenolic profiles, and ARC of Lalshak leaves under 50 and 100 mM salt concentrations were much higher in comparison to the control conditions. It could be used as a valuable food for human diets with health benefits. Salt-treated Lalshak leaves had abundant minerals, AsA, Folin-Ciocalteu reducing capacity, BC, TF, pigments, polyphenolic profiles, and ARC. Pigments, AsA, Folin-Ciocalteu reducing capacity, BC, TF, polyphenolic compounds, and ARC quench ROS; thus, Lalshak could be beneficial for human health via its potent antioxidant activities. Moreover, sodium chloride-enriched Lalshak provided outstanding quality in the final product in terms of nutrients, pigments, polyphenolic profiles, and ARC. We can cultivate it as an encouraging alternative vegetable in salt-prone zones of the world.